Menadione (2-methyl-1,4-naphtoquinone) is a synthetic member of the vitamin K family and is described as vitamin K3. It possesses the most simple structure among vitamin K family, with no aliphatic chain prosthetic group at position 3 of naphtoquinone skeleton (Fig.1). The best-known naturally occurring members of the vitamin K family are: vitamin K1 (phylloquinone),
which was found in many higher plants as well as algae (Thompson, 1971) and vitamin K2 (menaquinone) which is produced by intestinal bacteria from exogenous naphtoquinones (Seegers and Bang, 1967). Vitamin K3 treatment was applied in various types of rodent- and human-derived neoplastic cell lines in vitro, such as oral epidermal carcinoma, breast carcinoma, leukemia and hepatocellular carcinoma (HCC) cell lines (Chlebowski et al., 1985;
Markovits et al., 2003; Chen et al., 2002; Nutter et al., 1991; Lamson and Plaza, 2003; Verrax et al., 2003). Although the mechanisms of antitumor effects of vitamin K have been investigated intensively, they still remain unclear. Most of the data come from in vitro experiments and there are only small number of reports demonstrating in vivo antitumor activity of vitamin K3. There are suggested two mechanisms of antitumor effects of menadione. It can act as the oxidative stress inducer via redox-cycling of the quinone or it can arrest cell cycle at G1 phase (Kuriyama et al., 2005).
Historically, it was proposed that the menadione anti-cancer activity was due to oxidative stress via redox-cycling of the quinone to produce reactive oxygen species (ROS), such as the superoxide anion radical, hydroxyl radical, and hydrogen peroxide (Gant et al., 1988). Quinones can undergo either one-electron reduction, producing semiquinone radicals, or two-electron reduction, resulting in hydroquinones (Fig.2). The cytotoxicity of menadione may depend on direct arylation of nucleophiles such as glutathione and initiation one- or two-electron redox cycling (Lamson and Plaza, 2003). Redox cycling is defined as the ability to elicit a disproportionate NAD(P)H oxidation or oxygen utilization in such a biological system when compared with the quantity of quinone present, and involves the transfer of one electron to oxygen from the semiquinone intermediate. Redox cycling, together with the generation of reactive oxygen species and the subsequent oxidative stress induced, has been proposed as the mechanism by which quinones may cause toxicity and subsequently apoptosis (Gant et al., 1988; Criddle et al., 2006). The cytotoxicity of menadione displayed by redox cycling results
Fig.1. Menadione (Vitamin K3)
in thiols depletion accompanied by GSSG formation, NADPH oxidation and perturbation of calcium ion homeostasis (Thor et al., 1982).
Menadione possesses the ability to directly arylate thiols, depleting in this way the pool of glutathione and sulfhydryl-containing proteins, which comprises another aspect of an oxidative mechanism (Nishikawa et al., 1995). Menadione reacting directly with nucleophiles such as GSH by Michael addition (Nickerson et al., 1963) results in oxygen consumption, H2O2 and GSSG formation as well as production of a menadione-GSH conjunate (Ross et al., 1985; Dimonte et al., 1984). The decrease of sulfhydryl groups in treated cells suggests that vitamin K3 might also decrease the activities of other critical sulfhydryl-containing enzymes such as protein tyrosine phosphatases as well as p34Cdc2 protein associated with cell growth (Juan et al., 1996). There is another possibility that menadione inhibits glutathione reductase (GR) (Bellomo et al., 1987), what may prevent the reduction of GSSG to GSH.
Reduced glutathione (GSH), a tripeptide (γ-L-glutamyl-L-cysteine-glycine), is an important intracellular redox buffer that exists as a reduced predominant form, as a disulfide form (GSSG) or as mixed disulfide (GSSR) with protein thiols (Deneke and Fanburg, 1989).
It is the most important antioxidant agent in the cells where it is present in mM concentrations. During the oxidative stress, GSH is oxidized instead of lipids, proteins or nucleic acids. Glutathione reductase participate in regeneration of GSH and it uses the NADPH produced in PPP as a reducing cofactor. The ability of the cell to diminish oxidative stress may be impared due to decreased potential of the PPP (Riganti et al., 2004). The GSH/GSSG ratio reflects the redox status within the cell (Cotgreave and Gerdes, 1998) and this is responsible for the regulation of pro-inflammatory genes (Rahman and MacNee, 2000).
A decreasing GSH/GSSG ratio inhibits the binding activity of NF-κB in endothelial and alveolar epithelial cells (Chen et al., 2000; Haddad et al., 2000). Significant reduction of intracellular GSH levels is reflected by a low GSH/GSSG ratio, a possible result of the formation of glutathionyl adducts between GSH and quinone which is also due to the glutathione S-transferases (GST), which marks these adducts for export from the cell (Awad et al., 2002). These compounds alkylate thiol groups, mainly through the formation of thioether derivatives of cysteine (Bolton et al., 1997).
Menadione-induced oxidative stress is associated with a perturbation of intracellular Ca2+ homeostasis (Bellomo et al., 1982; Thor et al., 1982). During the metabolism of menadione, Ca2+ is released from intracellular stores, and the ability of mitochondria and microsomes to sequester Ca2+ is impaired. In addition, the metabolism of menadione results in the inhibition of plasma membrane Ca2+-ATPase activity. The critical involvement of protein
sulfhydryl group oxidation in the menadione-mediated inhibition of the Ca2+-ATPase is suggested by the finding that GSH was able to restore the impaired ATPase activity (Nicotera et al., 1985). NAD(P)H oxidation by menadione, results in inhibition of aerobic glycolysis (Rossi and Zatti, 1964), stimulation of pentose phosphate pathway activity (Rossi and Zatti, 1964; Rossi and Zoppi, 1966), and depletion of the mitochondrial ATP pools (Bellomo et al., 1982). Further studies revealed that menadione induced depletion of NAD(P)H results in depletion of mitochondrial ATP and loss of control of the flux of ionized calcium across mitochondrial and cellular membranes (Bellomo et al., 1982). Loss of control of ionized calcium flux, a process influenced by reduced glutathione, may be one mechanism by which depletion of reduced gluthatione pools result in cytotoxicity (Bellomo et al., 1982).
2.1. Non-oxidative model of menadione antitumor activity
Apart from oxidative mechanism, menadione exerts antitumor effects by affecting the key molecules of G1 phase cell cycle regulation (Kuriyama et al., 2005). Cell cycle molecules
Fig.2. Redox cycling of menadione. (adapted and modified from Lamson and Plaza, 2003) Menadione
1e -1e
-2e -NAD(P)/H+
NADP+
NQO1
1e
-O2
O2O ●
Semiquinone Intermediate form
Hydroquinone Reduced Menadione
1e -O2
O2O ●
play essential roles in carcinogenesis and tumor development. G1 phase-related molecules are especially important because they are required for the entry into the cell cycle from the quiescent state. Cell cycle molecules are divided into 3 groups, namely cyclins, Cdks and Cdk inhibitors. Among cyclins, the D-type (D1, D2 and D3), specifically cyclin D1, serve as a critical regulator of the cell cycle (Hanahan and Weinberg, 2000). Cyclin D1 forms complexes with Cdk4 and these proteins are responsible for driving cell cycle from G1 to S phase. Cdk inhibitors, especially of the INK family, are G1-phase specific and consist of p15INK4b, p16INK4a, p18INK4c and p19INK4d. These inhibitors are active only on Cdk4- or Cdk6-containing complexes. Moreover, binding of the INK family proteins to Cdk4 or to Cdk6 is independent of cyclin D (Chan et al., 1995; Hirai et al., 1995; Serrano et al., 1993). Because members of this family are known to bind and inhibit Cdk4 and Cdk6 without affecting other Cdks (Sherr, 1996), they are G1 phase-specific. p16INK4a inhibits the turnover of cell cycle and makes cells stay at G1 phase.
Retinoblastoma (Rb) is the ultimate substrate of cyclin D1/Cdk4 and cyclin D1/Cdk6 complexes in the pathway leading to transition from G1 to S phase (Sherr, 1996). Rb protein controls gene expression mediated by a family of heterodimeric transcriptional regulators, described as E2Fs, which can transactivate genes which products are essential for S phase entry (Kuriyama et al., 2005). In its phosphorylated form, Rb protein binds to a subset of E2F complexes, converting them to repressors that constrain expression of E2F target genes.
Phosphorylatin of Rb protein frees these E2Fs, enabling them to transactivate the same genes, a process initially triggered by cyclin D1/Cdk4 and cyclin D1/Cdk6 complexes, and then accelerated by cyclin E/Cdk2 complexes (Kuriyama et al., 2005). Kuriyama et al. (2005) demonstrated in vivo that vitamin K3 exert antitumor actions by regulating the expression of cell cycle-related molecules. Their research on human hepatocellular carcinoma (HCC) cells revealed that menadione reduced the mRNA expression of Cdk4, but not that of cyclin D1 and increased mRNA expression of p16INK4a and Rb. Therefore, reduced cyclin D1/Cdk4 kinase activities induced by vitamin K3 cause reduced proliferative activity of HCC cell, resulting in retarded HCC development (Kuriyama et al., 2005). On the other hand, increased p16INK4a expression in HCC tumors suppressed cyclin D1/Cdk4 and cyclin D1/Cdk6 kinase activities, resulting in in vivo antitumor effects of menadione on HCC. This case suggest that menadione antitumor activity is at least in part due to cell cycle arrest at G1 phase of HCC cells (Kuriyama et al., 2005). Jamison et al. (2004) demonstrated that human bladder tumor cells exposed to combined treatment of vitamin K3 and C also results in cell cycle arrest. In addition, cells that were in G1 phase at the time of vitamin treatment are arrested in G1, while
those which have passed the G1 checkpoint progress through the S phase and become arrested in G2/M. The G2/M arrest is believed to depend on the regulation of cyclin B1 and p34cdc2 (Clopton and Saltman, 1995).
2.2. Synergistic antitumor chemotherapeutic action of MEN
Menadione was found to act as antitumor drug synergistic with cisplatin, 5-fluorouracil (5-FU), dacarbazine, and bleomycin in human oral epidermoid carcinoma cell culture.
Synergistic action between vitamin K3 and doxorubicin, vinblastine, and 5-FU was also demonstrated in nasopharyngeal carcinoma cells (Liao et al., 2000). Synergistic effect of menadione and mitomycin C treatment was observed in lung cancer (Tetef et al., 1995) and advanced gastrointestinal cancers (Tetef et al., 1995).
Many clinical useful antitumor agents have a quinone group in their structure.
Menadione possesses a broad spectrum of antitumor activity including multidrug-resistant human cancer cell lines. This compound may not exhibit serious toxic side effects in humans, in particular, cardiac toxicity, such as seen after in patients treated with doxorubicin, and may be a useful candidate in combination chemotherapy (Nutter et al., 1991; Thompson, 1971).
Synergistic cytotoxic activity of the combination of vitamins C and K3 possesses the features of cell death which is described as autoschizis (from the Greek autos, self, and schizein, to split, as defined by Gilloteaux et al., 1998). Ultrastructural studies of vitamin-treated tumor cells undergoing autoschizis revealed exaggerated membrane damage and an enucleation process in which the pericaria separate from the main cytoplasmic body by self-excision. These self-excisions continue until all that remained is an intact nucleus surrounded by a narrow rim of cytoplasm that contains damaged organelles, including SER, RER, mitochondria, membrane whorls, lysosomes, and lipid droplets (Gilloteaux et al., 1998). In the self-excising cells, all organelles surround the nucleus as a tight mass of membranes, vacuolated mitochondria, and mitochondria with intramatrical deposits, as well as apparently intact pieces of RER cisterns (Gilloteaux et al., 2001). The nucleus exhibits nucleolar segregation and chromatin decondensation followed by nuclear karryohexis and karyolysis (Gilloteaux et al., 1998; Gilloteaux et al., 1998; Gilloteaux et al., 2001; Gilloteaux et al., 2001; Jamison et al., 2002).